1. Field of the Invention
The invention relates to soluble methane monooxygenase (sMMO) constitutive mutant strains of methanotrophic bacteria capable of degrading chlorinated hydrocarbons in the presence of copper. Also disclosed are methods of selecting methanotrophic bacteria to provide mutants deficient in particulate methane monooxidase (pMMO) activity with retained soluble MMO (sMMO) activity.
2. Description of Related Art
Chlorinated aliphatic solvents represent a major class of pollutants due to their widespread distribution in the environment and their toxic or carcinogenic effects. They are produced in large quantities and are the most frequently detected organic pollutants found in drinking water from groundwater sources (Fan, 1988). Trichloroethylene (TCE), a common contaminant, is a known carcinogen in animals and a suspected carcinogen in humans (Infante and Tsongas, 1987). It is also an especially recalcitrant compound in aquifers, and undergoes biodegradation only slowly (Parsons, et al., 1984).
Chloroform is another ubiquitous contaminant, particularly prevalent as a byproduct in water and wastewater treatment processes where chlorine is used as a disinfectant. Trace amounts may often be found in drinking water sources in areas close to commercial operations.
Methanotrophic bacteria are unusually active in degrading chlorinated hydrocarbons such as chloroform and trichloroethylene (TCE), (Wilson and Wilson, 1985). Methanotrophs are capable of growth with methane as their sole carbon and energy source. Although chloroform and TCE cannot serve as a sole carbon source, it is cometabolized by these organisms under aerobic conditions, removing as much as 99% of the TCE at concentrations as high as 50 mg/liter (Fliermans, et al., 1988).
Well over a hundred strains of methanotrophic bacteria have been isolated. Of these, Methylosinus trichosporium OB3b degrades TCE at a rate at least one order of magnitude faster than other pure or mixed cultures (Hansen, et al., 1990).
The key enzyme responsible for this high rate of TCE removal is a methane monooxygenase (MMO), the enzyme responsible for the first step in methane oxidation by these microbes. The bacterial methane monooxygenases have broad substrate specificity, and the specificity of the soluble form (sMMO) of the enzyme generally is broader than the particulate form (pMMO) found associated with stacked internal membranes. The sMMO form is less frequently found in methanotrophic bacteria, but has been reported in Methylosinus trichosporium, Methylococcus capsulatus, and Methylobacterium sp. strain CRL26 (Patel, et al., 1982). Following incubation with whole cells of M. trichosporium OB3b, or purified sMMO with added NADH reductant, TCE is more than 90% mineralized to CO.sub.2 and Cl.sup.- (Oldenhuis, et al. 1989).
A major problem in the use of M. trichosporium in bioremediation projects lies in its expression of MMO. Of the two forms of this enzyme, pMMO is normally expressed, while sMMO is expressed only when cells are grown under copper limitation (Burrows, et al., 1989; Stanley, et al., 1983). Unfortunately, the levels of copper necessary to down-regulate the expression of sMMO are quite low, on the order of about 1 .mu.M, which is within the range of copper ion levels found in groundwater (Forstner and Wittman, 1979).
Despite the knowledge that sMMO is required to metabolize TCE and related chlorinated hydrocarbons, there has been no success in developing or isolating a M. trichosporium capable of metabolizing TCE and related compounds in the presence of copper ion. Copper is ubiquitous in most water sources and particularly in polluted water; economically practical means of removal are not presently available.